Dupont C, Broyart J P, Broer Y, Chenut B, Laburthe M, Rosselin G
J Clin Invest. 1981 Mar;67(3):742-52. doi: 10.1172/JCI110091.
An EDTA procedure was used to prepare isolated epithelial cells of human gallbladder devoid of endogenous vasoactive intestinal peptide (VIP) as measured by radioimmunoassay. Specific binding sites for VIP were characterized in these cells. At 37 degrees C, the binding of (125)I-labeled VIP reached a peak within 20 min and then declined rapidly. At 15 degrees C, binding was stable between 90 and 180 min of incubation. Binding of the labeled peptide was inhibited by concentrations of native VIP of 30 pM-0.1 muM. Half-maximal inhibition was observed at 2 nM. Scatchard analysis indicated two functionally independent classes of receptor sites: 62,000 high affinity sites/cell with a dissociation constant (K(d)) of 1.3 nM, and 510,000 low affinity sites/cell with a K(d) of 16.2 nM. Secretin inhibited tracer binding but with a 1,000 times lower potency than native VIP. VIP strongly stimulated adenosine 3':5' monophosphate (cyclic AMP) production in human gallbladder epithelial cells. At 37 degrees C, 0.1 nM and 10 nM VIP raised cyclic AMP levels 44 and 100 times above the basal level, respectively. Maximal values remained constant between 60 and 90 min at 15 degrees C. The importance of the VIP-induced cyclic AMP rise was related, at least in part, to a low phosphodiesterase activity in human gallbladder epithelial cells. At equilibrium, during a 60-min incubation at 15 degrees C, cyclic AMP production was noted at concentrations of VIP as low as 3 pM. Maximal and half-maximal stimulations were observed at 10 nM and 0.2 nM VIP, respectively. Secretin also stimulated cyclic AMP production but with a 10,000 lower potency than VIP. In the guinea pig, VIP and secretin were equipotent stimulators of cyclic AMP in gallbladder epithelial cells. This particular feature was shown to be due to receptors specific for each peptide that were present in these cells.
采用乙二胺四乙酸（EDTA）法制备人胆囊分离上皮细胞，通过放射免疫测定法检测发现其不含内源性血管活性肠肽（VIP）。对这些细胞中VIP的特异性结合位点进行了表征。在37℃时，125I标记的VIP的结合在20分钟内达到峰值，然后迅速下降。在15℃时，孵育90至180分钟期间结合稳定。天然VIP浓度为30 pM - 0.1 μM时可抑制标记肽的结合。在2 nM时观察到半数抑制。Scatchard分析表明存在两类功能独立的受体位点：每细胞62,000个高亲和力位点，解离常数（Kd）为1.3 nM；每细胞510,000个低亲和力位点，Kd为16.2 nM。促胰液素抑制示踪剂结合，但效力比天然VIP低1000倍。VIP强烈刺激人胆囊上皮细胞中3':5'-环磷酸腺苷（环磷酸腺苷）的产生。在37℃时，0.1 nM和10 nM的VIP分别使环磷酸腺苷水平比基础水平提高44倍和100倍。在15℃时，最大值在60至90分钟之间保持恒定。VIP诱导的环磷酸腺苷升高的重要性至少部分与人类胆囊上皮细胞中低磷酸二酯酶活性有关。在15℃孵育60分钟达到平衡时，在低至3 pM的VIP浓度下即可观察到环磷酸腺苷的产生。分别在10 nM和0.2 nM的VIP时观察到最大刺激和半数最大刺激。促胰液素也刺激环磷酸腺苷的产生，但效力比VIP低10,000倍。在豚鼠中，VIP和促胰液素是胆囊上皮细胞中环磷酸腺苷的等效刺激剂。这一特殊特征表明是由于这些细胞中存在每种肽的特异性受体。
